Topic 7 - modern genetics Flashcards
what doe PCR stand for?
polymerase chain reaction
what is a PCR used for?
To amplify DNA (make lots of copies of the sample)
What are the steps of a PCR?
denaturation:
heat DNA to 95 degrees
this splits strands by breaking h bonds
use enzyme from taq (can withstand heat)
annealing:
cool DNA to 55 degrees, primers bind to strands
extension:
heat to 72 degrees
add one polymerase strand to synthesise DNA from where the primer attached
cycle is repeated around 30 times to give enough DNA. sample can be used in DNA sequencing and DNA profiling.
what machine does a PCR?
thermocycler
what is a genome?
total genetic makeup of an organism
what are introns/exons
introns- nucleotide sequences that DON’T code for a gene
exons- nucleotide sequences DO code for a gene
why is a pH buffer used in PCR?
to keep the pH constant and maintain optimum pH level for enzyme activity
what is an intron?
DNA that does not code for a protein
within introns , there are satellites (STRs) - what are these?
satellites are found within introns , they are short sequences of DNA that are repeated many times.
what are the two types of satellites (STRs) ?
micro satellites- 2-6 bases repeated 5-100 times
mini satellites 10-100 bases, repeated 50-several hundred times
How do satellites (STRs) determine how closely related individuals are?
in a homologous pair of chromosomes, the same satellite is found at the same location - the thing that makes us unique is how often this is repeated.
The more closely related, the more similar the patternss in the size of satellites.
What are the stages of DNA profiling?
PCR happens previously.
- RESTRICTION ENDONUCLEASE ENZYMES
used to cut DNA as they are made to have a specific active site which fits onto the specific intron where it wants to cut - EITHER SIDE OF SATELLITE IS CUT
- GEL ELECTROPHORESIS
- SOUTHERN BLOTTING
alkaline buffer added (denatures DNA so it becomes single stranded)
Nylon filler placed on gel- this draws the DNA fragments to it leaving them as ‘blots’ attached. - ADD GENE PROBES
Gene probes are short single strand of DNA that have sequences complementary t sequences being sought.
Probes have fluorescent marker attached
bind with complementary DNA strands in a process called - hybridisation - GRAPH THE DATE COLLECTED FROM THE PROBE
What is the name of the process when cells become specialised?
differentiation
How do cells become specialised?
the genes we don’t want are switched off.
This means transcription doesn’t happen so RNA is not made , so gene is not translated.
what are house keeping proteins?
housekeeping proteins are proteins present in most cells , for example ATP synthase
you can compare between cells by comparing the RNAs (they all have same DNA)
what is a regulatory sequence and where is it found?
a regulatory sequence determines whether a gene is expressed or not -> these are found in our ‘junk DNA’ which consists of non-coding introns
regulatory sequences are either promoter or enhancer regions
a regulatory sequence can be either a promotor or enhancer region
what does a promotor region do?
initiate transcription by enabling RNA polymerase to bind to the region they regulate
a regulatory sequence can be either a promotor or enhancer region
what does an enhancer region do?
makes the gene more/less accessible by opening the chromatin structure (by loosening or tightening DNA around histome (when tightened, no gene)
what is a transcription factor?
Transcription factors are proteins that bind to DNA. They either stimulate/prevent transcription of the gene.
They can either be:
promotor sequences- enable binding of RNA polymerase and therefore enable transcription
enhancer sequences- regulate DNA activity by changing chromatin structure. If structure open, gene is expressed. If closed, gene inactivity.
explain how splicing happens
splicing is post-transcriptional modification of the mRNA. Helps explain how cells produce more proteins than they have genes, because it results in different products from a single gene.
happens during transcription.
full section of DNA copied onto mRNA (now known as pre-mRNA)
splice the pre-mRNA to remove introns (and some exons) - leaving only useful sections of exons
this is done by an enzyme called a spliceosome (this cuts, sticks and glues DNA)
now mature/functional mRNA can leave nucleus
What are isoforms?
isoforms are different arrangements of exons- therefore one gene can produce several polypeptides because different exons can be removed during splicing.
define epigenetics
the study of how your genome is expressed
explain DNA methylation
happens to adenine or cytosine (cytosine in vertebrates)
done by enzyme methyltransferase
- CH3 (methyl) group added to the pyrimidine (in nitrogenous base)
- this can only happen at a CpG region (when a C and G are next to each other)
-occurs at promotor regions and prevents activation of RNA polymerase , so the gene is not transcribed
how can methylation change your gene over time?
although your genome (DNA) does not change , your epigenome does vary.
more or less (demethylation) methyl groups are added during different stages of life.
they are hereditary
your epigenome can also be affected by factors of the environment , such as diet and smoking
explain histone modification
histone and DNA is chromatin.
the histone can either be:
acetylated:
a CH3 joins to lycine or leucine
acetyl group transferred to molecules (donated by acetyl coenzyme A)
activates chromatin, allowing genes to be transcribed
methylated:
a CH3 joins to lycine or leucine
this tightens DNA, having an inhibitory effect
stem cells
are capable of self renewal and of producing new cells that differentiate into other specialised types of cell
define totipotent , pluripotent and multipotent cells
all are unspecialised and can keep dividing.
toti - can differentiate into any cell type, including placenta
pluri- can differentiate into any cell , apart from placenta
multi- can only differentiate into cells of a certain tissue
pluripotent cells are affected by _________________ within blastocyst ?
location
what are induced pluripotent stem cells? (iPS cells)
adult stem cells taken from the body that have been reprogrammed to become pluripotent again.
1) fibroblasts (connective tissue) taken from skin samples.
2) transcription factors added to activate specific genes.
3) iPS behave similarly to embryonic stem cells.
how is therapeutic cloning carried out?
remove nucleus from both body cell and ovum
combine body cell nucleus into ovum
multiply to make more pluripotent cells
separate and allow them to differentiate
transport into patient with no rejection issues.
How is recombinant DNA made?
(DNA from more than one organism)
1) isolate the gene
method 1:
use restriction endonucleases
which cuts a specific base sequence of DNA. Leaves ‘sticky ends’, exposed complementary bases that can clip back together
method 2:
use reverse transcriptase to turn mRNA into cDNA (complimentary DNA)
DNA polymerase used to make the other strand of DNA
2) cut plasmid with same restriction endonuclease to leave complementary sticky ends.
3) join plasmid and gene with DNA ligase
4)reincorporate plasmid into host nucleus
NEXT (after either method)
plasmids are vector for gene to be inserted into another organism
plasmid’s sticky ends are complimentary to sticky ends of DNA
plasmid is cut with restriction endonuclease enzyme and DNA ligase sticks DNA into plasmid by joining up sugar phosphate backbone.
Reincorporate plasmid into host cell nucleus.
What is the method of identifying recombiant DNA?
REPLICA PLATING
plasmid contains marker gene (these could be fluorescent, antibiotic resistant or have specific requirement for nutrient)
grow bacteria on plate that contains everything (master plate)
sample bacteria using velvet
grow again without the factor the plasmid should contain
compare to the original plate to compare which colonies are modified
What are the uses of recombiant DNA?
identify what a particular gene does
create many copies of a particular protein
insert useful genes into an organism so it expresses a particular protein.
What are the four methods of inserting DNA into cells?
1) gene gun
coat recombinant DNA in heavy metal and fire through membrane.
2)use virus
insert weakened form of virus into recombinant DNA cells (endocytosis)
3)liposome wrapping
lipid droplets fuse with cell surface membrane and can therefore enter cell
4)microinjection
insert DNA directly into nucleus
What are knockout mice?
knockout mice are mice with one or more genes silenced , via the insertion of a similar gene which make the original gene impossible to read. they can be used to investigate gene function or understand disease.
1) insert gene similar to one being investigated
2)this stops original gene being transcribed
3)insert into embryonic stem cell (zygote) and then into a host mother.
4)chimeric mice are born- they have both versions of gene
5) cross breeding with normal mouse leads to homozygous mice which only have the knockout gene.
define transgenic plants
these are plants which contain genetic material from an unrelated organism.
How are soya beans transgenic?
Linoleic acid is replaced by oleic acid which oxidises less easily and therefore doesn’t go off as quickly.
What can genetically modified plants be used for?
pesticide protection
changing nutrient value of plants
increase crop yield
What are the objections to genetically modified crops?
favours richer, more developed countries
unnatural
What is DNA sequencing ?
DNA sequencing is used to predict amino acid sequence of proteins and determine links to genetic conditions.
What is DNA profiling and how does it work?
DNA profiling is used to identify criminals and test paternity.
1) fragments are cut with restriction endonuclease enzymes either side of the satellite.
2) fragments separated and visualised using gel electrophoresis.
3) southern blotting
alkaline buffer added which denatures the DNA so it becomes single stranded. Nylon filter draws the DNA fragments leaving them as ‘blots’ attached.
4) gene probes (short single strands of DNA that have sequences complementary to sequences being sought) have fluorescent markers and bind with complementary DNA strands in a process called hybridisation.
5) graph the data collected from the probe.
What are the 3 ways gene expression can be changed by epigenetics?
DNA methylation
Histone modification
Non-coding RNA
Why are epigenetic modifications important?
ensure cell differentiation
evaluate induced pluripotent stem cells
+ overcomes ethical issues as a zygote is not made
- have shown tendency to be cancerous
What are gene markers?
gene markers are used to show where a foreign gene has been inserted. Fluorescence, antibiotic resistance combined with replica plating can be used as gene markers.
What is replica plating?
used to access if gene was inserted correctly when creating recombinant DNA.
plasmid contains marker gene.
bacteria grown on master plate.
sample bacteria using velvet.
grow again without the factor the plasmid should contain, compare to the original plate to identify which colonies are modified.
